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Abstract

BACKGROUND: While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively).

METHODS AND RESULTS: The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin.

CONCLUSIONS: The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.